Poster U20 - MiR-145: A KEY REGULATOR OF BONE MARROW STROMAL CELL DIFFERENTIATION

Introduction: MicroRNA (miRNAs) are small, 18–22 nucleotides long, non-coding RNA molecules that have emerged as potent regulators of gene expression, playing crucial roles in stem cell fate determination. Mature miRNA strands bind to the RNA-Induced Silencing Complex (RISC), altering protein production by preventing translation of messenger RNA (mRNA). MiR-145 is a specific miRNA that inhibits osteoblast differentiation and the production of osteoprotegerin (OPG). There is evidence that miR-145 influences the communication between osteoblasts and osteoclasts by regulating production of OPG, which competitively inhibits binding of RANK ligand (RANKL) to the RANK receptor on osteoclasts, consequently impacting bone resorption. We hypothesized that miR-145 and its inhibitor can be used as a potent modulator of osteoblast differentiation of bone marrow stromal cells (MSCs) to control bone homeostasis.

Materials and

Methods: MicroRNA mimics and inhibitors were provided as pre-miRNA in stable hairpin loop strands (ThermoFisher Scientific). hMSCs were cultured on 24 well culture plates and grown to confluence with growth media or osteogenic differentiation media for 10 days. On day 11, cells were treated with miR-145 mimic or inhibitor at high (40nM) and low (20nM) concentrations, delivered via a lipophilic transfection agent, lipofectamine, for 48 hours. On day 13, media were exchanged for fresh media for 24 hours. On day 14, the cells were harvested. Outcome measurements assessed markers of osteoblast differentiation including alkaline phosphatase (ALP) specific activity, osteocalcin (OCN), osteopontin (OPN), osteoprotegerin (OPG), and vascular endothelial growth factor (VEGF) production, as well as total DNA content.

Results, Conclusions, and Discussions: Higher concentrations of miR-145 mimic (miR-145m) resulted in increased ALP activity, and decreased VEGF, OCN, and OPG production indicating decreased osteoblast differentiation compared to the osteogenic media control. Total DNA content and OPN were not affected by mimic treatment. Conversely, higher concentrations of miR-145 inhibitor (miR-145i) resulted in decreased DNA content and ALP activity, and increased production of OCN, OPN, & OPG indicating enhanced osteoblast differentiation compared to the osteogenic media control. There were no observed difference in VEGF production. Interestingly, the effects of both the mimic and inhibitor seemed to only impact bMSCs actively differentiating into osteoblasts, as there were minimal observed differences between cells cultured in growth media and treated with the miRNA mimic or inhibitor compared to the growth media control.
The data demonstrate a dose-dependent effect of miR-145m and miR-145i on osteoblast differentiation. Treating MSCs with the mimic resulted in decreased production of osteoblast markers, with the exception of ALP activity, which increased. Treatment with miR-145i increased production of osteoblast markers with the exception of ALP activity, which decreased. Collectively this could indicate mir-145 delays osteoblast differentiation as increased ALP is an early stage marker of osteoblast differentiation. These results suggest that miR-145 is important in regulating osteoblast differentiation, and consequently, could be a key contributor in regulating bone formation. The study provides valuable insights into the mechanisms underlying miR-145’s regulation of osteoblast differentiation and its potential therapeutic applications in modulating bone homeostasis. Future directions of this study seek to encapsulate miR-145 in lipid nanoparticles for targeted local delivery and test the viability of miR-145 in modulating bone formation in vivo through intraosseous injection into the bone marrow.

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